The optimal fraction of cholesterol in lung surfactants remains controversial; the role of cholesterol in lung surfactant function is still unknown. Small amounts of cholesterol, when added to DPPC or to clinical lung surfactants, reduce the surface shear viscosity by orders of magnitude, without altering the minimum surface tension. We have found that cholesterol separates into a disordered interphase that reduces the line tension between semi-crystalline DPPC-rich domains, which, in turn, dramatically alters domain morphology. This hypothesize that this interphase lubricates flow, causing the reductions in monolayer viscosity and elasticity, thereby enhancing the surfactant's ability to flow and cover the interface. At higher cholesterol concentrations, we hypothesize that the interphase properties eliminate the monolayer necessary monolayer cohesion so that collapse occurs at higher surface tensions. These observations suggest an optimal cholesterol content exists for a replacement lung surfactant. We will determine this optimal cholesterol content by measuring the shear viscosity and elasticity of clinical and model lung surfactants as a function of cholesterol composition using macro- and micro- rheology instruments unique to our laboratory. These mechanical properties will be correlated with isotherms, fluorescence and atomic force microscopy, and grazing incidence synchrotron X-ray diffraction to determine how cholesterol alters the molecular packing of lung surfactant lipids, which determines the mechanical properties of monolayers necessary for low surface tensions and rapid respreading and adsorption. Our goal is to determine the physiologically optimal viscosity and elasticity for rapid spreading and low surface tension and how best to achieve this optimum by controlling the cholesterol, lipid and protein fractions of a synthetic replacement lung surfactant for respiratory distress syndrome. In addition to an optimal composition, sufficient surfactant must be adsorbed to the interface from the alveolar fluid during the respiratory cycle. The lung surfactant specific proteins SP-A, B and C, along with lipids such as phosphatidylglycerol and cholesterol, are hypothesized to enhance exchange between surfactant in the subphase and the interface. However, little quantitative evidence for specific lipid and/or protein exchange exists. Also unknown is the surface pressures at what adsorption occurs, or if adsorption occurs during compression or expansion of the interface. To address this hypothesis, we will map out the three- dimensional distribution of lung surfactant components from the interface to the subphase using vertical and horizontal optical sectioning with a confocal microscope and multiple fluorescent dyes. We expect that SP-A, B and C promote adsorption; however, we do not know if specific lipids or proteins are adsorbed preferentially to the interface to optimize the monolayer composition that collapses at high surface pressures. Native SP-A, B and C will be compared to peptide mimics to evaluate the efficacy of the peptides.